Genetic regulatory modules are responsible for many critical cellular functions such as cell cycle control, DNA damage response, and signal transduction. These modules appear to have evolved high-level properties such as robustness (maintenance of function despite mutations) and evolvability (acquisition of new functionality due to mutations). Although such properties are fundamental for understanding evolutionary change at the cellular level, they are often difficult to define precisely, and measure. This project takes advantage of an alternative approach: the design and construction of simple synthetic modules. These are regulatory networks built from scratch out of a small number of well-characterized genetic elements. They can implement a wide spectrum of biological functionality in cells using very simple components. Most importantly, having been designed without regard to biological utility, and not subject to selection or optimization by evolution, they allow quantitative definition and measurement of robustness and evolvability in modules that have not already been selected for those very characteristics. The work will begin with a previously described library of synthetic modules that make binary logical decisions, based on two chemical inputs. Each synthetic module will be mutagenized at specific sites, to make a combinatorial library of specific changes to its component sequence elements. Thus, the quantitative parameters that control promoter strength, translation efficiency, and DNA-binding affinity will be systematically varied. The resulting changes in module functionality will be measured. This data is sufficient to define a measure of robustness and evolvability, and determine the relationship between these two properties. Modeling and simulation efforts, tightly coupled to the experimental system, complement the experiments. The long-term goal is a detailed understanding of how the regulatory structure of a transcription regulatory module determines both its function, and the ability to maintain or change that function, during evolution.